Usable nitrogen is the major limiting nutrient in crop plant growth. Plants derive most of their nutrients including nitrogen from the soil through uptake in the root system. Although most of the nitrogen in the soil is in the form of ammonium ions which is rapidly converted to usable nitrates by bacteria in the soil, the harvesting of plants results in a steady decrease of nitrogen from the soil. Unless the soil is augmented with nitrogen-containing compounds, the soil becomes depleted of usable nitrogen and only atmospheric nitrogen remains.
Legumes, unlike other higher plants, are able through a symbiotic relationship with bacteria to utilize atmospheric nitrogen in the soil. The bacteria, Rhizobia, infect leguminous seedlings and induce nodulation, the end result being the presence within the root system of nodules which contain the rhizobial bacteroids. Once within the root system, the bacteroids are able to xe2x80x9cfixxe2x80x9d atmospheric nitrogen into organic compounds the legumes can use. In exchange for the conversion of atmospheric nitrogen, the plants provide the bacteroids with carbon-containing compounds, other nutrients, and a protective environment.
Although the xe2x80x9cfixedxe2x80x9d nitrogen is used throughout the plant in the growth and development of its organs and tissues, much of the usable nitrogen remains within the nodules of the roots. This empirical finding has led to the practice of crop rotation wherein a non-leguminous plant, i.e., corn, is grown and harvested and then the field is sown with a legume, such as alfalfa. After harvest of the legume, the remaining roots are plowed under and thus, usable nitrogen is returned to the soil for the sowing of the non-leguminous crop.
The legumes recognize the rhizobial bacteria through a lectin-carbohydrate interaction. Within the root system, the plants contain lectins that bind to specific carbohydrates found on the Rhizobium cell wall. This interaction is very specific; with each plant recognizing and being infected by one rhizobial strain.
In addition to their involvement in recognition of rhizobial bacteria, oligosaccharide signaling events play important roles in the regulation of plant development, defense, and other interactions of plants with the environment (Ryan, C. A. and Farmer, E. E. Annu. Rev. Plant Physiol. Plant Mol. Bio. 42:651-674 (1991); Cote, F. and Hahn, M. G. Plant Mol. Biol 26:1379-1411 (1994); Denarie, I. et al. Annu. Rev. Biochem. 65:503-535 (1996)). Although the structures of some of these oligosaccharides have been characterized, little is known about the plant receptors for these signals, nor the mechanism(s) by which these signals are transduced.
Previously, a root lectin, NBP46 (formerly called DB46), was isolated from young Dolichos biflorus root extracts. NBP46 is a 46 kDa protein that was isolated by affinity chromatography on hog gastric mucin blood group A+H substance conjugated to Sepharose (Quinn, J. M. and Etzler, M. E. Arch. Biochem. Biophys. 258:535-544 (1987)).
Identification and characterization of protein and the genes that encode them is important to modulation of oligosaccharide signaling in plants. For instance, a transgenic non-leguminous plant containing a factor that allows rhizobial bacteria to infect the plant and fix nitrogen would lessen the need for the addition of nitrogen-containing fertilizer to soil and preclude the necessity of crop rotation in nitrogen-depleted fields. This would lead to higher yields of crop plants in areas of the world where the soil has been overplanted and replenishment of the depleted soil with usable nitrogen. The present invention addresses these and other needs.
This invention provides for the isolation and cloning of the cDNA of NBP46 (SEQ ID NO:1), which encodes NBP46, a Nod factor binding lectin. Nod factors are carbohydrates on the surface of Rhizobium which bind to lectins on the surface of leguminous plant organs and can initiate nodulation of the root system by the plants. The NBP46 gene encodes a polypeptide of between 50 and 560 amino acids, more preferably 462 amino acids (SEQ ID NO:2).
In a preferred embodiment, the NBP46 coding sequence is operably linked to a plant specific promoter, more preferably a root specific promoter, such as the NBP46 promoter (SEQ ID NO:3).
In another embodiment, an expression cassette comprising the NBP46 gene is introduced into a transgenic plant. In a preferred embodiment, the expression of NBP46 by the transgenic plant confers to the plant the ability to bind to rhizobial bacteria and utilize atmospheric nitrogen. In a particularly preferred embodiment, the expression of NBP46 confers to the plant the ability to catalyze the hydrolysis of the phosphoanhydride bonds of di- and tri-phosphates, leading to greater availability of nutrients to the plant.
In a further embodiment of the instant invention, methods of modulating the rhizobial interactions and in the phosphatase activity in plants by the introduction of an expression cassette comprising NBP46 are disclosed.